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Derek Lowe's commentary on drug discovery and the pharma industry. An editorially independent blog from the publishers of Science Translational Medicine. All content is Derek’s own, and he does not in any way speak for his employer.

Animal Testing

A Toxicological Flag

Here’s a caution from a new paper out of Manchester. The group had been synthesizing inhibitors of PARG (poly-ADP ribose glycohydrolase), an enzyme involved in DNA repair. The general chemotype is shown at right, but there are a number of variations. That fluorine is a new addition, though. The corresponding cyclopropylmethyl series came from their earlier paper, but they found that the series wasn’t particularly stable to metabolic oxidation. The major metabolites proved to be the primary sulfonamides in each case, suggesting that oxidation of the cyclopropylmethyl itself (which would be most likely at the methyl carbon) was the problem. Indeed, fluorination as shown increased the microsomal stability substantially (difluorination decreased the affinity for the PARG target itself, and deuteration, interestingly, had no real effect).

So far, so good. But trouble hit when the compounds were dosed in mice – there were obvious signs of toxicity, and deaths at the higher doses. This had never been seen with the earlier compounds, and dosing a fluoromethyl analog that had significantly reduced PARG potency showed the same effects. The trouble was noted at around the five-hour point, and only after oral dosing. Necropsy showed obvious signs of liver damage, which fits with that observation as well.

That all adds up to off-target tox brought on by metabolic activation of the fluoromethylcyclopropyl group. What the active species might be is not clear yet, nor is the specific liver target. But these observations alone are enough to flag that modification as a serious potential problem, which is the whole reason this new paper was published. Monofluoroalkyls can be a little odd – terminal monofluoroalkanes get cleaved down to fluoroacetic acid, for example, which is toxicological bad news. And fluorobenzyls (monofluoromethyl aryls) are reactive as electrophiles, despite the fluorocarbon reputation for stability. But this appears to be something different from either of those cases.

It’s true that this may vary according to the rest of the molecule, but just seeing it show up this strongly in one series is cause for concern. And since cyclopropyl groups and fluorinated methyls are both pretty common motifs in medicinal chemistry, it’s important to realize that the combination could be bad news. A quick Reaxys search of the fluoromethylcyclopropylsulfonamide side chain gave 183 hits, but those are all from two patents filed by the Manchester group themselves. But if you search for the corresponding amide, you also pick up a couple of compounds from Gilead as IRAK-4 inhibitors. The amine is a perfectly reasonable-looking building block, and it would be a good thing if it did not spread through the med-chem literature without people knowing that there may be a red flag attached to it.

Long story short: in some situations cancer cells are slightly more sensitive to DNA damage than normal cells.
Partly this is because cancer cells divide faster than your normal cells, and usually have lost a lot of checks to their replication. Thus cancer cells accumulate DNA damage and die, whereas normal cells take things slow, repair their DNA and survive; or don’t replicate at all and stay put.
In some situations, cancer cells might also have lost some DNA repair pathways, whereas normal cells have more redundancy at their disposal. Therefore a normal cell will repair their DNA with a pathway that we have not inhibited, and the cancer cell dies.

Or so goes the theory. In practice, things are messy, so we just try and see what happens?

More than just in theory as PARP inhibitors have shown some exceptionally good results in BRCA deficient tumors through synthetic lethality, the concept that shutting down to components of homologous recombination DNA repair causes cell death. There are now 4 (I think) approved PARP inhibitors.

I’m not as familiar with PARG but I’ll assume they work in the same pathway and the thought is the same.

PARG likely regulates DNA repair by enzymatically-degrading the poly ADP ribose synthesized by PARP 1 an enzyme that is, as pointed out, also involved in DNA repair. PARP 1 has been shown to be an efficacious target in, as also pointed out, BRCA-deficient tumors.

Fluorines are pretty terrible leaving groups and the C-F bond is a very strong bond. Seems like sort of a bad combination of a nitrogen and the fluoromethyl that could result in a particularly reactive cyclopropyl ring.

Cyclopropylcarbinyl radical or cation as an intermediate? The bond strength and the leaving group ability depend in part on the stability of the resulting radical or cation; if memory serves, the cyclopropylcarbinyl versions are considerably more stable than the classical localized radical and cation.

cyclopropylcarbinyl radical formed by CYP mediated oxidation will pop open the cycloproane to give the sulfonamido substituted but-2-eneyl system with an allylic terminal radical on one end and the fluoromethyl on the other end. Radical is quenched one way or another (time dependent CYP inhibitor?) In any event, the examine system can hydrolase to the alpha fluormethyl ketone.

Meant to say: cyclopropylcarbinyl radical formed by CYP mediated oxidation will pop open the cyclopropane to give the 1-fluoro-2-sulfonamido substituted but-1-enyl system with a terminal radical at the 4-position. Radical is quenched one way or another (time dependent CYP inhibitor?) In any event, the examine system can hydrolase to the alpha fluormethyl ketone.

Possibly some neighboring group participation with displacement of fluorine linked to formation of cyclobutyl cation. Sulfonamide then deprotonates to give what would be a very electrophilic imine (alternatively sulfonamide deprotonation could be synchronized with displacement of fluorine). On a semi-related note the weaker activity of the CF2 and CF3 analogs may be due to effect on sulfonamide NH rather than steric.

The fluorine would make chasing metabolism by NMR the obvious step – fluorine NMR on unpurified samples to see what modifications had happened near the fluorine. Modern spectrometers can detect less than a microgramme.

Do you realize that in addition to fluoridating water, why, there are studies underway to fluoridate salt, flour, fruit juices, soup, sugar, milk, PARG INHIBITORS, ice cream? Children’s ice cream!…You know when fluoridation began?…1946. 1946, How does that coincide with your post-war Commie conspiracy, huh? It’s incredibly obvious, isn’t it? A foreign substance is introduced into our precious bodily fluids without the knowledge of the individual, and certainly without any choice.
That’s the way your hard-core Commie works. First PARG Inhibitors, tomorrow the World!

The hydrolysis metabolite of the compound which is 1-(fluoromethyl)cyclopropanol will be unstable and will undergo rearrangement to 1-fluorobutan-2-one. The last one may be responsible for the toxicity. For, example, fluoroacetone is a highly toxic compound.